The clinical importance of non-HLA specific antibodies in kidney transplantation

The clinical importance of non-HLA specific antibodies in kidney

transplantation

Ayeda Almahri

2015

Laboratory for Transplantation and Regenerative Medicine, Department of Clinical Chemistry and Transfusion Medicine

Institute of Biomedicine, the Sahlgrenska Academy

ISBN -– 978-91-628-9311-8

Printed by Ale Tryckteam, Bohus 2015

To my parents, brothers and sisters, your support have sustained me throughout my whole life.

To my dear husband Humaid Almahri, you made me stronger with your words and advice.

Love you all!

ISBN -– 978-91-628-9311-8

Printed by Ale Tryckteam, Bohus 2015

To my parents, brothers and sisters, your support have sustained me throughout my whole life.

To my dear husband Humaid Almahri, you made me stronger with your words and advice.

Love you all!

The clinical importance of non-HLA specific antibodies in kidney transplantation

Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine

Sahlgrenska Academy at University of Gothenburg

Abstract

The clinical significance of human leukocyte antigen (HLA) antibodies (Abs) for hyperacute, acute and chronic antibody-mediated rejection (AMR) of kidney allografts has been clearly demonstrated. AMR occurs in the absence of donor- reactive HLA Abs. It is not known how common the problem of AMR by non-HLA Abs is because of lack of suitable assays for their detection. It is believed that the non- HLA Ab population, although heterogenic, is likely to target antigens on donor organ endothelial cells (ECs). We have been involved in the clinical introduction of a flow cytometric (FC) crossmatch (XM) test that permits the detection of Abs reactive with endothelial precursor cells (EPC) isolated from donor peripheral blood. In this context the EPCs may function as surrogates for mature vascular ECs.

The work in this thesis describes the adaptation of the EPCXM to detection of complement-fixing HLA and non-HLA Abs using complement fragment-specific antibodies and flow cytometry, describes the outcome of the EPCXM in relation to the conventional lymphocyte XM (LXM), degree of HLA sensitization and transplantation outcome in patients evaluated for living donor (LD) kidney transplantation (Tx), and assesses the long-term renal graft function in patients with a positive EPCXM pre-transplant.

In the first paper, we investigated whether EPCs could be used for detection of complement-fixing Abs and if complement factor and IgG deposition on co-purified T and B cells correlated to the outcome of the T- and B-cell complement-dependent

cytotoxicity (CDC) XM. Incubation of EPCs with HLA Ab-positive serum samples resulted in deposition of complement factors C3c and C3d, but not C1q nor C4d, on EPCs and co-purified lymphocytes. The amount of C3c deposition and IgG binding on EPCs and T cells, but not B cells, correlated. The specificity and sensitivity for C3d deposition on co-purified T cells vs the T CDC assay were 69% and 72%, while for B cells the sensitivity was considerably lower. In the second paper, we show that 32% of the LD patients had IgG and/or IgM-binding donor EPCs in their pre-Tx sera.

Twenty-five percent of the patients were EPCXM IgM+. Of the patients with negative LXM tests, 25% had EPC Abs mainly of IgM class not reactive with HLA. There was no difference in rejection frequency or serum creatinine levels between the EPCXM positive and negative groups, which is in contrast to earlier published results.

However, the clinical protocols used in the second paper included Ab pre-Tx treatments such as B cell depletion and Ab removal. The pre-Tx EPCXM positive group had significantly more patients with delayed graft function. In the manuscript we show that the difference in serum creatinine and glomerular filtration rates observed between EPCXM positive and negative groups at three and six months post- Tx disappears hereafter and during the four-year follow-up.

The detection of complement factors on EPCs and lymphocytes by flow cytometry allowing detection of complement-fixing non-HLA and HLA Abs widens the diagnostic repertoire that can be offered patients undergoing kidney transplantation and should thereby improve their clinical management. Prospective studies with appropriate control groups are needed to establish whether pre-treatments aiming at removing anti-EC Abs, as detected by the EPCXM pre-Tx, have a beneficial effect on short- and long-term graft survival.

ISBN –

978-91-628-9311-8

The clinical importance of non-HLA specific antibodies in kidney transplantation

Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine

Sahlgrenska Academy at University of Gothenburg

Abstract

The clinical significance of human leukocyte antigen (HLA) antibodies (Abs) for hyperacute, acute and chronic antibody-mediated rejection (AMR) of kidney allografts has been clearly demonstrated. AMR occurs in the absence of donor- reactive HLA Abs. It is not known how common the problem of AMR by non-HLA Abs is because of lack of suitable assays for their detection. It is believed that the non- HLA Ab population, although heterogenic, is likely to target antigens on donor organ endothelial cells (ECs). We have been involved in the clinical introduction of a flow cytometric (FC) crossmatch (XM) test that permits the detection of Abs reactive with endothelial precursor cells (EPC) isolated from donor peripheral blood. In this context the EPCs may function as surrogates for mature vascular ECs.

The work in this thesis describes the adaptation of the EPCXM to detection of complement-fixing HLA and non-HLA Abs using complement fragment-specific antibodies and flow cytometry, describes the outcome of the EPCXM in relation to the conventional lymphocyte XM (LXM), degree of HLA sensitization and transplantation outcome in patients evaluated for living donor (LD) kidney transplantation (Tx), and assesses the long-term renal graft function in patients with a positive EPCXM pre-transplant.

In the first paper, we investigated whether EPCs could be used for detection of complement-fixing Abs and if complement factor and IgG deposition on co-purified T and B cells correlated to the outcome of the T- and B-cell complement-dependent

cytotoxicity (CDC) XM. Incubation of EPCs with HLA Ab-positive serum samples resulted in deposition of complement factors C3c and C3d, but not C1q nor C4d, on EPCs and co-purified lymphocytes. The amount of C3c deposition and IgG binding on EPCs and T cells, but not B cells, correlated. The specificity and sensitivity for C3d deposition on co-purified T cells vs the T CDC assay were 69% and 72%, while for B cells the sensitivity was considerably lower. In the second paper, we show that 32% of the LD patients had IgG and/or IgM-binding donor EPCs in their pre-Tx sera.

Twenty-five percent of the patients were EPCXM IgM+. Of the patients with negative LXM tests, 25% had EPC Abs mainly of IgM class not reactive with HLA. There was no difference in rejection frequency or serum creatinine levels between the EPCXM positive and negative groups, which is in contrast to earlier published results.

However, the clinical protocols used in the second paper included Ab pre-Tx treatments such as B cell depletion and Ab removal. The pre-Tx EPCXM positive group had significantly more patients with delayed graft function. In the manuscript we show that the difference in serum creatinine and glomerular filtration rates observed between EPCXM positive and negative groups at three and six months post- Tx disappears hereafter and during the four-year follow-up.

The detection of complement factors on EPCs and lymphocytes by flow cytometry allowing detection of complement-fixing non-HLA and HLA Abs widens the diagnostic repertoire that can be offered patients undergoing kidney transplantation and should thereby improve their clinical management. Prospective studies with appropriate control groups are needed to establish whether pre-treatments aiming at removing anti-EC Abs, as detected by the EPCXM pre-Tx, have a beneficial effect on short- and long-term graft survival.

ISBN –

978-91-628-9311-8

Populärvetenskaplig sammanfattning

Bakgrund: Patienter med kraftigt nedsatt njurfunktion behöver dialys för att överleva, ofta flera gånger i veckan. Genom njurtransplantation förbättras patientens livskvalitet och hen kan i allmänhet gå tillbaka till ett fullt yrkesverksamt liv. En fruktad komplikation vid njurtransplantation är avstötningsreaktionen, d.v.s. den reaktion där patientens immunsystem försöker stöta bort njuren. Denna reaktion orsakas bl.a. av antikroppar som känner igen de s.k. transplantationsantigenerna (HLA), vilket är strukturer som finns på alla kroppens celler och som oftast skiljer sig åt mellan olika individer. I en del fall kan antikroppar mot andra strukturer, vilka ofta sitter på kärlens insida på de s.k. endotelcellerna, än HLA orsaka antikroppsförmedlad avstötning.

Syfte: Arbetet i denna avhandling har syftat till att förfina en ny metod för att hos patienter inför njurtransplantation upptäcka antikroppar riktade mot den donerade njurens endotelceller, och att undersöka hur dessa antikroppar korrelerar till risken för avstötning och nedsatt funktion hos den transplanterade njuren.

Material och metoder: Patienternas HLA typ bestämdes med genetiska metoder (PCR) och om de hade HLA antikroppar eller inte avgjordes med cellbaserade metoder och solid fasmetoder. Förenlighet mellan patient och donator testades i s.k.

korstester i vilka eventuella antikroppar i patientserum får binda till donatorns

lymfocyter (en celltyp i blod som bär HLA antigen). Avläsningen av korstesten sker i

mikroskop eller med s.k. flödescytometri. Den senare är en känslig metod för att

undersöka om patientantikroppar bundit till donatorcellerna. Vidare användes och

vidareutvecklades en ny korstestmetod som möjliggör detektion av antikroppar mot

endotelcellsliknande celler från donatorn.

Populärvetenskaplig sammanfattning

Bakgrund: Patienter med kraftigt nedsatt njurfunktion behöver dialys för att överleva, ofta flera gånger i veckan. Genom njurtransplantation förbättras patientens livskvalitet och hen kan i allmänhet gå tillbaka till ett fullt yrkesverksamt liv. En fruktad komplikation vid njurtransplantation är avstötningsreaktionen, d.v.s. den reaktion där patientens immunsystem försöker stöta bort njuren. Denna reaktion orsakas bl.a. av antikroppar som känner igen de s.k. transplantationsantigenerna (HLA), vilket är strukturer som finns på alla kroppens celler och som oftast skiljer sig åt mellan olika individer. I en del fall kan antikroppar mot andra strukturer, vilka ofta sitter på kärlens insida på de s.k. endotelcellerna, än HLA orsaka antikroppsförmedlad avstötning.

Syfte: Arbetet i denna avhandling har syftat till att förfina en ny metod för att hos patienter inför njurtransplantation upptäcka antikroppar riktade mot den donerade njurens endotelceller, och att undersöka hur dessa antikroppar korrelerar till risken för avstötning och nedsatt funktion hos den transplanterade njuren.

Material och metoder: Patienternas HLA typ bestämdes med genetiska metoder (PCR) och om de hade HLA antikroppar eller inte avgjordes med cellbaserade metoder och solid fasmetoder. Förenlighet mellan patient och donator testades i s.k.

korstester i vilka eventuella antikroppar i patientserum får binda till donatorns

lymfocyter (en celltyp i blod som bär HLA antigen). Avläsningen av korstesten sker i

mikroskop eller med s.k. flödescytometri. Den senare är en känslig metod för att

undersöka om patientantikroppar bundit till donatorcellerna. Vidare användes och

vidareutvecklades en ny korstestmetod som möjliggör detektion av antikroppar mot

endotelcellsliknande celler från donatorn.

Resultat och diskussion: I det första arbetet vidareutvecklade vi det flödescytometriska korstestet som möjliggör identifiering av antikroppar mot donatorns endotelceller till att också identifiera de antikroppar som kan aktivera komplement. Denna typ av antikroppar är mer potenta och utgör en större risk för avstötning. I det andra arbetet visade vi att patienter med antikroppar detekterade i endotelcellskorstestet i högre grad hade njurar som kom igång senare efter transplantationen och t.o.m. ibland förlorades. I manuskriptet har vi följt njurtransplanterade patienter över tid för att se hur njurfunktionen hos de patienter som hade endotelcellsantikroppar utvecklades över tid. Den skillnad i njurfunktion vi såg tre och sex månader efter transplantationen mellan grupper med och utan endotelcellsantikroppar försvann från ett år efter transplantation och fortsatt under den fyraåriga uppföljningen.

Sammanfattning: Vi har vidareutvecklat ett test som möjliggör identifiering av en antikroppspopulation som tidigare ej kunnat identifieras och som kan bidra till försämrad funktion, och i värsta fall avstötning, av njurtransplantat.

List of publications

This thesis is based on the following studies, referred to in the text by their Roman numerals.

I. Detection of complement-fixing and non-fixing antibodies specific for endothelial precursor cells and lymphocytes using flow cytometry. Ayeda AlMahri, Jan Holgersson, Mats Alheim. Tissue Antigens, 2012, 80, 404–415

II. The outcome of the endothelial precursor cell crossmatch test in lymphocyte crossmatch positive and negative patients evaluated for living donor kidney transplantation. Mats Alheim, Ayeda AlMahri, Jakob Nilsson, Gunnar Tydén, Jan Holgersson. Human Immunology, 2013, 74, 1437–1444

III. A pre-transplant positive endothelial precursor cell crossmatch does not imply

reduced long-term kidney graft function. Markus Gäbel, Ayeda AlMahri,

Lennart Rydberg, Jan Holgersson, Michael E. Breimer. (Manuscript).

Resultat och diskussion: I det första arbetet vidareutvecklade vi det flödescytometriska korstestet som möjliggör identifiering av antikroppar mot donatorns endotelceller till att också identifiera de antikroppar som kan aktivera komplement. Denna typ av antikroppar är mer potenta och utgör en större risk för avstötning. I det andra arbetet visade vi att patienter med antikroppar detekterade i endotelcellskorstestet i högre grad hade njurar som kom igång senare efter transplantationen och t.o.m. ibland förlorades. I manuskriptet har vi följt njurtransplanterade patienter över tid för att se hur njurfunktionen hos de patienter som hade endotelcellsantikroppar utvecklades över tid. Den skillnad i njurfunktion vi såg tre och sex månader efter transplantationen mellan grupper med och utan endotelcellsantikroppar försvann från ett år efter transplantation och fortsatt under den fyraåriga uppföljningen.

Sammanfattning: Vi har vidareutvecklat ett test som möjliggör identifiering av en antikroppspopulation som tidigare ej kunnat identifieras och som kan bidra till försämrad funktion, och i värsta fall avstötning, av njurtransplantat.

List of publications

This thesis is based on the following studies, referred to in the text by their Roman numerals.

I. Detection of complement-fixing and non-fixing antibodies specific for endothelial precursor cells and lymphocytes using flow cytometry. Ayeda AlMahri, Jan Holgersson, Mats Alheim. Tissue Antigens, 2012, 80, 404–415

II. The outcome of the endothelial precursor cell crossmatch test in lymphocyte crossmatch positive and negative patients evaluated for living donor kidney transplantation. Mats Alheim, Ayeda AlMahri, Jakob Nilsson, Gunnar Tydén, Jan Holgersson. Human Immunology, 2013, 74, 1437–1444

III. A pre-transplant positive endothelial precursor cell crossmatch does not imply

reduced long-term kidney graft function. Markus Gäbel, Ayeda AlMahri,

Lennart Rydberg, Jan Holgersson, Michael E. Breimer. (Manuscript).

Table of Contents

Introduction ... 15

I. Kidney Transplantation ... 15

II. Renal allograft rejection ... 16

Acute cellular rejection ... 19

Antibody-mediated rejection ... 19

Chronic rejection ... 21

III. Mechanisms of allorecognition ... 22

IV. The HLA system ... 24

V. HLA antibodies ... 25

VI. The complement system ... 29

VII. Alloantigens beside HLA (non-HLA) including endothelial cell antigens………... .30

VIII. Immunological evaluation ... 31

HLA typing ... 31

HLA antibody detection and specificity determination ... 33

The crossmatch test ... 35

Complement-dependent cytotoxicity crossmatch………...…36

The flow cytometric crossmatch………...……….36

Testing for Abs against non-HLA including anti-endothelial cell antibodies ... 37

Aims of the thesis ... 38

Table of Contents

Introduction ... 15

I. Kidney Transplantation ... 15

II. Renal allograft rejection ... 16

Acute cellular rejection ... 19

Antibody-mediated rejection ... 19

Chronic rejection ... 21

III. Mechanisms of allorecognition ... 22

IV. The HLA system ... 24

V. HLA antibodies ... 25

VI. The complement system ... 29

VII. Alloantigens beside HLA (non-HLA) including endothelial cell antigens………... .30

VIII. Immunological evaluation ... 31

HLA typing ... 31

HLA antibody detection and specificity determination ... 33

The crossmatch test ... 35

Complement-dependent cytotoxicity crossmatch………...…36

The flow cytometric crossmatch………...……….36

Testing for Abs against non-HLA including anti-endothelial cell antibodies ... 37

Aims of the thesis ... 38

Methodological considerations ... 39

Endothelial precursor cell crossmatch assay ... 39

Flow cytometric complement deposition assay ... 42

Results and discussion ... 45

Concluding remarks ... 56

Future perspectives ... 57

Acknowledgements ... 58

References ... 60

List of Abbreviations Abs Antibodies

AECA Anti-endothelial cell antibodies AMR Antibody-mediated rejection ATG Anti-thymocyte globulin ATN Acute tubular necrosis AT1R Angiotensin Type 1 receptor CAN Chronic allograft nephropathy CDC Complement-dependent cytotoxicity DSA Donor-specific antibodies

EC Endothelial cell

ELISA Enzyme-linked immunosorbent assay ESRD End stage renal disease

FACS Fluorescence-activated cell sorting HLA Human leukocyte antigen

KTx Kidney transplantation LD Living donor

mGFR Measured glomerular filtration rate MHC Major histocompatibility complex MMF Mycophenolate mofetil

PBMC Peripheral blood mononuclear cells PCR Polymerase chain reaction

PRA Panel-reactive antibodies SCr Serum creatinine

SSO Sequence-specific oligonucleotides

SSP Sequence-specific primer

Methodological considerations ... 39

Endothelial precursor cell crossmatch assay ... 39

Flow cytometric complement deposition assay ... 42

Results and discussion ... 45

Concluding remarks ... 56

Future perspectives ... 57

Acknowledgements ... 58

References ... 60

List of Abbreviations Abs Antibodies

AECA Anti-endothelial cell antibodies AMR Antibody-mediated rejection ATG Anti-thymocyte globulin ATN Acute tubular necrosis AT1R Angiotensin Type 1 receptor CAN Chronic allograft nephropathy CDC Complement-dependent cytotoxicity DSA Donor-specific antibodies

EC Endothelial cell

ELISA Enzyme-linked immunosorbent assay ESRD End stage renal disease

FACS Fluorescence-activated cell sorting HLA Human leukocyte antigen

KTx Kidney transplantation LD Living donor

mGFR Measured glomerular filtration rate MHC Major histocompatibility complex MMF Mycophenolate mofetil

PBMC Peripheral blood mononuclear cells PCR Polymerase chain reaction

PRA Panel-reactive antibodies SCr Serum creatinine

SSO Sequence-specific oligonucleotides

SSP Sequence-specific primer

TCMR T-cell-mediated rejection Tx Transplantation

XM Crossmatch

Introduction

I. Kidney Transplantation

Organ transplantation (Tx) is performed in order to replace a diseased or damaged organ with a healthy one. Patients with end-stage renal diseases (ESRD) are treated either by dialysis or kidney transplantation. ESRD occurs when both kidneys are no longer functional because of defective kidney filtering capacity leading to accumulation of waste products, perturbed salt balance and hormonal deregulation [1]. Symptoms of ESRD may remain mild or absent until kidney function drops to less than 20% of normal [2]. Symptoms can be significant and include, but are not limited to, weight loss, nausea or vomiting, general malaise, fatigue, headache, hiccups, itching, decreased urination, easy bruising or bleeding, lethargy, difficulty breathing, and seizures. Causes of ESRD include diabetes, high blood pressure and atherosclerosis, autoimmune diseases (e.g. lupus), genetic disorders (like polycystic kidney disease), infections, post-renal obstruction of the urinary tract, and exposure to toxic substances (e.g. antibiotics, chemotherapy, dyes used for contrast in radio imaging, analgesics, fungal toxins) [3].

Kidney transplantation is done in order to correct ESRD and in the majority of cases kidney transplantation allows the patient to return to a normal life and full time work [4]. The kidney donor can be living (e.g. a parent, sibling or a child of the recipient, a friend or spouse), or a deceased donor [5, 6].

In 1954 the first successful kidney transplantation was performed using an identical

twin brother as donor. The graft functioned well without immunosuppressive drugs

for 9 years until relapse of the underlying disease [7]. Complications sometimes seen

following kidney transplantation include those related to the surgical procedure and

TCMR T-cell-mediated rejection Tx Transplantation

XM Crossmatch

Introduction

I. Kidney Transplantation

Organ transplantation (Tx) is performed in order to replace a diseased or damaged organ with a healthy one. Patients with end-stage renal diseases (ESRD) are treated either by dialysis or kidney transplantation. ESRD occurs when both kidneys are no longer functional because of defective kidney filtering capacity leading to accumulation of waste products, perturbed salt balance and hormonal deregulation [1]. Symptoms of ESRD may remain mild or absent until kidney function drops to less than 20% of normal [2]. Symptoms can be significant and include, but are not limited to, weight loss, nausea or vomiting, general malaise, fatigue, headache, hiccups, itching, decreased urination, easy bruising or bleeding, lethargy, difficulty breathing, and seizures. Causes of ESRD include diabetes, high blood pressure and atherosclerosis, autoimmune diseases (e.g. lupus), genetic disorders (like polycystic kidney disease), infections, post-renal obstruction of the urinary tract, and exposure to toxic substances (e.g. antibiotics, chemotherapy, dyes used for contrast in radio imaging, analgesics, fungal toxins) [3].

Kidney transplantation is done in order to correct ESRD and in the majority of cases kidney transplantation allows the patient to return to a normal life and full time work [4]. The kidney donor can be living (e.g. a parent, sibling or a child of the recipient, a friend or spouse), or a deceased donor [5, 6].

In 1954 the first successful kidney transplantation was performed using an identical

twin brother as donor. The graft functioned well without immunosuppressive drugs

for 9 years until relapse of the underlying disease [7]. Complications sometimes seen

following kidney transplantation include those related to the surgical procedure and

secondary complications caused by the life-long immunosuppressive treatment. They can also be classified as short term (vascular thrombosis, narrowing of the renal artery, obstruction of the ureter, urine leakage, acute rejection) or long-term (chronic rejection and negative effects caused by the immunosuppression including diabetes, high blood pressure, cancer and infections) complications [8-10].

Despite an ever increasing success rate of kidney transplantation which to a large part can be ascribed ever better and more effective immunosuppressive drugs, there is still room for improvements.

II. Renal allograft rejection

Increased serum creatinine post-transplant may suggest allograft rejection, but other conditions such as surgical complications, infections and drug toxicity can impair renal graft function and lead to a rise in serum creatinine [11]. Histopathological assessment of biopsies taken from the transplanted kidney is key to the diagnosis, and the morphology may influence the choice of therapy and subsequent prognosis [12, 13]. Besides evaluation of the histology of the biopsy, detection and specificity determination of donor-specific antibodies is important in order to determine whether the rejection is predominantly T-cell or antibody-mediated. Subclinical rejections, i.e. rejections not associated with a rise in serum creatinine, may be evident only upon examination of the biopsy.

Renal allograft rejection can be divided into acute T-cell mediated rejection (ACR), acute antibody-mediated rejection (AMR) and chronic rejection (CR) based on biopsy morphology and the presence of donor-specific antibody [14, 15]. AMR can be either hyperacute (HAR) occurring within minutes to hours, or acute occurring within days to weeks after transplantation. Donor-specific HLA antibodies have also been

implicated as a pathogenic factor in CR, which sometimes occur years after

transplantation [16]. It should be emphasized though that many times a mixture of T-

cell and antibody-mediated pathology contribute to rejection and that acute and

chronic rejections may not be distinct events but rather represent a continuum of

events [17]. The type of rejection of renal grafts can also be classified according to the

histopathological picture found in the biopsy. This so called Banff classification (Table

1) of renal allograft biopsies grades the degree of interstitial infiltration of

mononuclear cells, the number of mononuclear cells per tubular cross section, and the

degree of arteritis in case of acute T-cell mediated rejection, and the degree of

interstitial fibrosis and tubular atrophy in case of chronic rejection [17]. Complement

factor C4d+ staining, the presence of circulating donor-specific antibodies (DSA) and

morphologic evidence of acute tissue injury such as acute tubular necrosis (ATN)-like

minimal inflammation, capillary and/or glomerular inflammation and/or

thrombosis, or transmural arteritis are diagnostic criteria for AMR [18, 19].

secondary complications caused by the life-long immunosuppressive treatment. They can also be classified as short term (vascular thrombosis, narrowing of the renal artery, obstruction of the ureter, urine leakage, acute rejection) or long-term (chronic rejection and negative effects caused by the immunosuppression including diabetes, high blood pressure, cancer and infections) complications [8-10].

Despite an ever increasing success rate of kidney transplantation which to a large part can be ascribed ever better and more effective immunosuppressive drugs, there is still room for improvements.

II. Renal allograft rejection

Increased serum creatinine post-transplant may suggest allograft rejection, but other conditions such as surgical complications, infections and drug toxicity can impair renal graft function and lead to a rise in serum creatinine [11]. Histopathological assessment of biopsies taken from the transplanted kidney is key to the diagnosis, and the morphology may influence the choice of therapy and subsequent prognosis [12, 13]. Besides evaluation of the histology of the biopsy, detection and specificity determination of donor-specific antibodies is important in order to determine whether the rejection is predominantly T-cell or antibody-mediated. Subclinical rejections, i.e. rejections not associated with a rise in serum creatinine, may be evident only upon examination of the biopsy.

Renal allograft rejection can be divided into acute T-cell mediated rejection (ACR), acute antibody-mediated rejection (AMR) and chronic rejection (CR) based on biopsy morphology and the presence of donor-specific antibody [14, 15]. AMR can be either hyperacute (HAR) occurring within minutes to hours, or acute occurring within days to weeks after transplantation. Donor-specific HLA antibodies have also been

implicated as a pathogenic factor in CR, which sometimes occur years after

transplantation [16]. It should be emphasized though that many times a mixture of T-

cell and antibody-mediated pathology contribute to rejection and that acute and

chronic rejections may not be distinct events but rather represent a continuum of

events [17]. The type of rejection of renal grafts can also be classified according to the

histopathological picture found in the biopsy. This so called Banff classification (Table

1) of renal allograft biopsies grades the degree of interstitial infiltration of

mononuclear cells, the number of mononuclear cells per tubular cross section, and the

degree of arteritis in case of acute T-cell mediated rejection, and the degree of

interstitial fibrosis and tubular atrophy in case of chronic rejection [17]. Complement

factor C4d+ staining, the presence of circulating donor-specific antibodies (DSA) and

morphologic evidence of acute tissue injury such as acute tubular necrosis (ATN)-like

minimal inflammation, capillary and/or glomerular inflammation and/or

thrombosis, or transmural arteritis are diagnostic criteria for AMR [18, 19].

Table 1 Banff 97 diagnostic categories for renal allograft biopsies—Banff’07 update Banff’s

classification Degree of rejection Characteristics and subtypes Normal

Antibody-mediated

rejection Acute AMR C4d+, DSA

Type I: ATN-like minimal inflammation Type II: capillary and/or glomerular inflammation

Type III: transmural arteritis

Chronic active AMR C4d+, DSA, glomerular double contours and/or peritubular capillary basement membrane multilayering and/or interstitial fibrosis/tubular atrophy and/or fibrous intimal thickening in arteries

Borderline ”Suspicious” TCMR Tubulitis (t1, t2 or t3) with interstitial infiltration (i0 or i1)

Interstitial infiltration (i2 or i3) with mild (t1) tubilitis

T-cell-mediated

rejection Acute TCMR Type IA: i2 or i3 and t2

Type IB: i2 or i3 and t3

Type IIA: mild-to-moderate intimal arteritis (v1)

Type IIB: severe intimal arteritis (v2) Type III: ”transmural” arteritis and/or

arterial fibrinoid change, necrosis and lymphocytic inflammation (v3) Chronic active TCMR Arterial intimal fibrosis with

mononuclear cell infiltration in fibrosis, formation of neo-intima

Interstitial fibrosis and tubular atrophy (IFTA)

Interstitial fibrosis and tubular atrophy Grade I: mild

Grade II: moderate Grade III: severe

Other Changes not due to

rejection Chronic/sclerosing allograft

nephropathy, recurrent diseases, toxic changes, and infection

Modified from Solez et al [19].

Acute cellular rejection

A rapid rise in serum creatinine may be caused by acute cellular rejection. Besides the increase in serum creatinine, patients may retain fluids, i.e. gain weight, develop fever and graft tenderness. The incidence of ACR is approximately 5-10% in the first year in unsensitized patients [20]. ACR is histologically characterized by an accumulation of mononuclear cells, mostly CD4+ and CD8+ T cells, in the intersititium, the tubules (causing tubulitis) and sometimes in the arteries (causing arteritis) [21, 22].

T cells cause cell damage by release of cytotoxic granules containing perforin and granzyme A and B, by engaging the Fas-FasL receptor pair and by releasing inflammatory cytokines (IFN-γ, TNF-α) and chemokines (CCL5/RANTES, CCL3/MIP-1) [23]. Tubulus infiltrating T cells and macrophages in tubulitis make tubular cells go into apoptosis as revealed by an increased number of TUNEL+ cells.

Subendothelial and intimal infiltration of T cells and macrophages is characteristic of endarteritis, a hallmark of ACR [24] .The latter is detected in 25-40% of renal biopsies taken on the suspicion of ACR and is rarely found in stable grafts [25]. At times, also glomerulitis is found in ACR cases.

Antibody-mediated rejection

Antibody-mediated rejection can be divided into hyperacute (HAR) and acute antibody-mediated rejection (AMR) depending on the kinetics of the rejection.

HAR is a very dramatic response that occurs immediately after transplantation

usually within the first hours and sometimes immediately after release of the vascular

clamps. It is caused by pre-existing host antibodies that bind to antigens, commonly

human leukocyte antigens (HLA) or blood group ABH antigens present on the graft

Table 1 Banff 97 diagnostic categories for renal allograft biopsies—Banff’07 update Banff’s

classification Degree of rejection Characteristics and subtypes Normal

Antibody-mediated

rejection Acute AMR C4d+, DSA

Type I: ATN-like minimal inflammation Type II: capillary and/or glomerular inflammation

Type III: transmural arteritis

Chronic active AMR C4d+, DSA, glomerular double contours and/or peritubular capillary basement membrane multilayering and/or interstitial fibrosis/tubular atrophy and/or fibrous intimal thickening in arteries

Borderline ”Suspicious” TCMR Tubulitis (t1, t2 or t3) with interstitial infiltration (i0 or i1)

Interstitial infiltration (i2 or i3) with mild (t1) tubilitis

T-cell-mediated

rejection Acute TCMR Type IA: i2 or i3 and t2

Type IB: i2 or i3 and t3

Type IIA: mild-to-moderate intimal arteritis (v1)

Type IIB: severe intimal arteritis (v2) Type III: ”transmural” arteritis and/or

arterial fibrinoid change, necrosis and lymphocytic inflammation (v3) Chronic active TCMR Arterial intimal fibrosis with

mononuclear cell infiltration in fibrosis, formation of neo-intima

Interstitial fibrosis and tubular atrophy (IFTA)

Interstitial fibrosis and tubular atrophy Grade I: mild

Grade II: moderate Grade III: severe

Other Changes not due to

rejection Chronic/sclerosing allograft

nephropathy, recurrent diseases, toxic changes, and infection

Modified from Solez et al [19].

Acute cellular rejection

A rapid rise in serum creatinine may be caused by acute cellular rejection. Besides the increase in serum creatinine, patients may retain fluids, i.e. gain weight, develop fever and graft tenderness. The incidence of ACR is approximately 5-10% in the first year in unsensitized patients [20]. ACR is histologically characterized by an accumulation of mononuclear cells, mostly CD4+ and CD8+ T cells, in the intersititium, the tubules (causing tubulitis) and sometimes in the arteries (causing arteritis) [21, 22].

T cells cause cell damage by release of cytotoxic granules containing perforin and granzyme A and B, by engaging the Fas-FasL receptor pair and by releasing inflammatory cytokines (IFN-γ, TNF-α) and chemokines (CCL5/RANTES, CCL3/MIP-1) [23]. Tubulus infiltrating T cells and macrophages in tubulitis make tubular cells go into apoptosis as revealed by an increased number of TUNEL+ cells.

Subendothelial and intimal infiltration of T cells and macrophages is characteristic of endarteritis, a hallmark of ACR [24] .The latter is detected in 25-40% of renal biopsies taken on the suspicion of ACR and is rarely found in stable grafts [25]. At times, also glomerulitis is found in ACR cases.

Antibody-mediated rejection

Antibody-mediated rejection can be divided into hyperacute (HAR) and acute antibody-mediated rejection (AMR) depending on the kinetics of the rejection.

HAR is a very dramatic response that occurs immediately after transplantation

usually within the first hours and sometimes immediately after release of the vascular

clamps. It is caused by pre-existing host antibodies that bind to antigens, commonly

human leukocyte antigens (HLA) or blood group ABH antigens present on the graft

endothelium [26, 27]. Following antibody binding, complement is fixed, activated and the membrane attack complex (C5b-C9) deposited on the cell surface causing cell lysis/necrosis [28]. Platelet aggregation and the formation of microthrombi contribute to cessation of blood flow, and endothelial cell retraction cause leakage of red cells and fluid out in the interstitial tissue. The kidney becomes cyanotic and swollen.

Grafts that have undergone HAR have to be removed and replaced with another graft. However, improvements in cross-matching techniques and specificity determinations of HLA antibodies have made HAR a rare event [29].

Acute AMR is caused by antibodies binding to donor HLA or non-HLA expressed on endothelial cells. It is characterized by a rapid rise in serum creatinine which may occur days to weeks or even years after transplantation. It is believed that antibodies may contribute to acute rejection episodes in at least 25% of the cases [20]. Antibodies may act in concert with T-cells in an otherwise predominant ACR or may act alone in an AMR without clear signs of ACR. In sensitized patients, i.e. in patients previously transplanted, transfused or with earlier pregnancies, and in patients with poor compliance the humoral component may be even more significant.

Antibodies bound to the endothelium will activate complement causing endothelial cell injury, release of von Willebrand factor (vWF) and surface expression of P- selectin, which promote platelet aggregation and the formation of microthrombi. In addition, cytokines (IL-1α, IL-8), chemokines (CCL2), and the chemoattractants, C3a and C5a, which cause leukocytes to adhere to glomeruli (glomerulitis) or to dilated peritubular capillaries (margination) will be released [28]. The complement factors C4d, which is also a histopathological biomarker for AMR (see below), and C5b, which initiates the assembly of the membrane-attack complex, causes localized endothelial necrosis and apoptosis. In severe cases microthrombi, with hemorrhage

and arterial wall necrosis and infarction, occurs [28]. In order to rescue such grafts early diagnosis and treatment are necessary.

Deposition of the complement factor C4d, an inactive fragment of C4b, in the majority of peritubular capillaries in a ring formed pattern is a diagnostic hallmark of AMR.

The recognition of the significance of C4d deposition and novel diagnostic tools for detection and specificity determination of DSA, have dramatically increased our ability to diagnose AMR [30]. Donor-specific class I or II Abs are present in around 90% of the patients with C4d deposition. In ABO incompatible transplantation C3d deposition is associated with acute inflammation, while C4d deposition in this patient group can be seen even in histologically normal grafts [31].

The ability of DSA to cause AMR is highly associated with its ability to fix complement. Thus, IgG3 and IgG1 DSA are more pathogenic than IgG2 and IgG4 DSA [32]. In this context novel diagnostic tools that enable identification of complement fixing DSA may become increasingly important [33].

Treatment options for AMR includes besides high dose steroids and proliferation inhibitors, removal of antibodies (plasmapheresis or immunoadsorption), immunoglobulin injections, B-cell depleting antibodies (anti-CD20), complement inhibitors such as the anti-C5 antibody (eculizumab) and proteasome inhibitors (bortezomib) [34, 35].

Chronic rejection

Cellular or humoral mechanisms or a combination of both may contribute to chronic

rejection (CR), which occurs months or years after transplantation. The morphologic

characteristics of chronic rejection can be seen in the glomeruli as glomerulopathy, in

turn recognized ultra structurally as duplication or multilamination of the glomerular

endothelium [26, 27]. Following antibody binding, complement is fixed, activated and the membrane attack complex (C5b-C9) deposited on the cell surface causing cell lysis/necrosis [28]. Platelet aggregation and the formation of microthrombi contribute to cessation of blood flow, and endothelial cell retraction cause leakage of red cells and fluid out in the interstitial tissue. The kidney becomes cyanotic and swollen.

Grafts that have undergone HAR have to be removed and replaced with another graft. However, improvements in cross-matching techniques and specificity determinations of HLA antibodies have made HAR a rare event [29].

Acute AMR is caused by antibodies binding to donor HLA or non-HLA expressed on endothelial cells. It is characterized by a rapid rise in serum creatinine which may occur days to weeks or even years after transplantation. It is believed that antibodies may contribute to acute rejection episodes in at least 25% of the cases [20]. Antibodies may act in concert with T-cells in an otherwise predominant ACR or may act alone in an AMR without clear signs of ACR. In sensitized patients, i.e. in patients previously transplanted, transfused or with earlier pregnancies, and in patients with poor compliance the humoral component may be even more significant.

Antibodies bound to the endothelium will activate complement causing endothelial cell injury, release of von Willebrand factor (vWF) and surface expression of P- selectin, which promote platelet aggregation and the formation of microthrombi. In addition, cytokines (IL-1α, IL-8), chemokines (CCL2), and the chemoattractants, C3a and C5a, which cause leukocytes to adhere to glomeruli (glomerulitis) or to dilated peritubular capillaries (margination) will be released [28]. The complement factors C4d, which is also a histopathological biomarker for AMR (see below), and C5b, which initiates the assembly of the membrane-attack complex, causes localized endothelial necrosis and apoptosis. In severe cases microthrombi, with hemorrhage

and arterial wall necrosis and infarction, occurs [28]. In order to rescue such grafts early diagnosis and treatment are necessary.

Deposition of the complement factor C4d, an inactive fragment of C4b, in the majority of peritubular capillaries in a ring formed pattern is a diagnostic hallmark of AMR.

The recognition of the significance of C4d deposition and novel diagnostic tools for detection and specificity determination of DSA, have dramatically increased our ability to diagnose AMR [30]. Donor-specific class I or II Abs are present in around 90% of the patients with C4d deposition. In ABO incompatible transplantation C3d deposition is associated with acute inflammation, while C4d deposition in this patient group can be seen even in histologically normal grafts [31].

The ability of DSA to cause AMR is highly associated with its ability to fix complement. Thus, IgG3 and IgG1 DSA are more pathogenic than IgG2 and IgG4 DSA [32]. In this context novel diagnostic tools that enable identification of complement fixing DSA may become increasingly important [33].

Treatment options for AMR includes besides high dose steroids and proliferation inhibitors, removal of antibodies (plasmapheresis or immunoadsorption), immunoglobulin injections, B-cell depleting antibodies (anti-CD20), complement inhibitors such as the anti-C5 antibody (eculizumab) and proteasome inhibitors (bortezomib) [34, 35].

Chronic rejection

Cellular or humoral mechanisms or a combination of both may contribute to chronic

rejection (CR), which occurs months or years after transplantation. The morphologic

characteristics of chronic rejection can be seen in the glomeruli as glomerulopathy, in

turn recognized ultra structurally as duplication or multilamination of the glomerular

basement membrane, and in the vessels as peritubular capillaropathy with features similar to those of the glomerulopathy and, in the arteries as transplant arteriopathy which is characterized by thickening of the arterial intima [36]. In addition, kidneys undergoing chronic rejection may develop interstitial fibrosis and tubular atrophy [37]. The majority of glomerulopathy cases are associated with HLA class II DSA in serum and between 30-50% of these have C4d deposition in the peritubular capillaries [28, 38]. When glomerulopathy is accompanied by DSA and C4d deposition it is diagnostic of a chronic humoral rejection (CHR) [36]. Early AMR in sensitized patients is a risk factor for later glomerulopathy and accumulation of mononuclear cells in peritubular capillaries as seen in CHR is also a risk factor for later graft failure [39]. Transplant arteriopathy may develop either as a consequence of a C4d+ or – CR.

In the latter case macrophages and CD3+ T cells may be seen in the neointima [20].

III. Mechanisms of allorecognition

The alloreactive immune response is initiated by T cells recognizing foreign HLA antigens, which are widely expressed on different cell types. The importance of HLA for allorecognition and rejection is reflected in the fact that grafts from HLA identical siblings have significantly longer survival times than grafts from HLA non-identical donors [24, 40]. Host T cells can directly recognize donor HLA on graft cells (direct pathway of antigen presentation) or following processing in host antigen-presenting cells (APC; indirect pathway of antigen presentation) [20]. The TCR on CD4+ T cells bind peptides presented by HLA class II antigens, while TCRs on CD8+ T cells bind peptides presented by HLA class I [41]. Besides engagement of the T cell receptor, so called costimulatory molecules on the T cell need to be engaged by their cognate ligands, e.g. CD28/CTLA4:CD80/CD86, CD40:CD154, ICOS:ICOSL, OX40:OX40L, and CD27:CD70, in order for the T cell to be activated [42, 43]. Novel

immunosuppressive drugs inhibiting costimulatory receptors, e.g. CTLA4Ig, are currently being explored for use in transplant patients.

Because costimulation is needed for T cell activation to occur, APC, especially dendritic cells (DC), carrying ligands for T cell costimulatory molecules are essential triggers of the alloresponse [42]. T cell activation by DC takes place in the regional lymph nodes and spleen following migration of DC there from the graft [44]. Both donor and host DC can initiate the alloresponse [45]. The latter DCs migrate into the graft from the circulation and once they have taken up antigen, they migrate via the lymph to the draining lymph node; a process guided by chemokines (CCL19/CCL21:CCR7) [46].

CD4+ T cells develop into helper T cells (T

cells) following activation. They help in the maturation of B cells into plasma cells (and production of DSA) and memory B cells as well as in the maturation of macrophages [47]. CD8+ T cells or cytotoxic T cells are important effector cells in ACR. They mediate cytotoxicity via release of cytotoxic granules containing perforin and granzyme A and B or via secretion of toxic cytokines such as TNF-α and β [48].

Cross-talk between the innate and adaptive immune systems is important for a potent

alloresponse to occur. The inflammatory reaction caused by ischemia and

reperfusion, and by the surgical trauma itself, potentiates the immune response by

recruiting immune cells including APC to the graft [49]. Further, increased expression

of ligands for toll-like receptors (TLR), damage-associated molecular-pattern (DAMP)

receptors and other innate inflammatory molecules promote maturation and

activation of dendritic cells [50, 51]. The complement system, in particular C3a and

C5a, can directly activate intra-graft T cells and antigen-presenting cells (APC) [52-

basement membrane, and in the vessels as peritubular capillaropathy with features similar to those of the glomerulopathy and, in the arteries as transplant arteriopathy which is characterized by thickening of the arterial intima [36]. In addition, kidneys undergoing chronic rejection may develop interstitial fibrosis and tubular atrophy [37]. The majority of glomerulopathy cases are associated with HLA class II DSA in serum and between 30-50% of these have C4d deposition in the peritubular capillaries [28, 38]. When glomerulopathy is accompanied by DSA and C4d deposition it is diagnostic of a chronic humoral rejection (CHR) [36]. Early AMR in sensitized patients is a risk factor for later glomerulopathy and accumulation of mononuclear cells in peritubular capillaries as seen in CHR is also a risk factor for later graft failure [39]. Transplant arteriopathy may develop either as a consequence of a C4d+ or – CR.

In the latter case macrophages and CD3+ T cells may be seen in the neointima [20].

III. Mechanisms of allorecognition

The alloreactive immune response is initiated by T cells recognizing foreign HLA antigens, which are widely expressed on different cell types. The importance of HLA for allorecognition and rejection is reflected in the fact that grafts from HLA identical siblings have significantly longer survival times than grafts from HLA non-identical donors [24, 40]. Host T cells can directly recognize donor HLA on graft cells (direct pathway of antigen presentation) or following processing in host antigen-presenting cells (APC; indirect pathway of antigen presentation) [20]. The TCR on CD4+ T cells bind peptides presented by HLA class II antigens, while TCRs on CD8+ T cells bind peptides presented by HLA class I [41]. Besides engagement of the T cell receptor, so called costimulatory molecules on the T cell need to be engaged by their cognate ligands, e.g. CD28/CTLA4:CD80/CD86, CD40:CD154, ICOS:ICOSL, OX40:OX40L, and CD27:CD70, in order for the T cell to be activated [42, 43]. Novel

immunosuppressive drugs inhibiting costimulatory receptors, e.g. CTLA4Ig, are currently being explored for use in transplant patients.

Because costimulation is needed for T cell activation to occur, APC, especially dendritic cells (DC), carrying ligands for T cell costimulatory molecules are essential triggers of the alloresponse [42]. T cell activation by DC takes place in the regional lymph nodes and spleen following migration of DC there from the graft [44]. Both donor and host DC can initiate the alloresponse [45]. The latter DCs migrate into the graft from the circulation and once they have taken up antigen, they migrate via the lymph to the draining lymph node; a process guided by chemokines (CCL19/CCL21:CCR7) [46].

CD4+ T cells develop into helper T cells (T

cells) following activation. They help in the maturation of B cells into plasma cells (and production of DSA) and memory B cells as well as in the maturation of macrophages [47]. CD8+ T cells or cytotoxic T cells are important effector cells in ACR. They mediate cytotoxicity via release of cytotoxic granules containing perforin and granzyme A and B or via secretion of toxic cytokines such as TNF-α and β [48].

Cross-talk between the innate and adaptive immune systems is important for a potent

alloresponse to occur. The inflammatory reaction caused by ischemia and

reperfusion, and by the surgical trauma itself, potentiates the immune response by

recruiting immune cells including APC to the graft [49]. Further, increased expression

of ligands for toll-like receptors (TLR), damage-associated molecular-pattern (DAMP)

receptors and other innate inflammatory molecules promote maturation and

activation of dendritic cells [50, 51]. The complement system, in particular C3a and

C5a, can directly activate intra-graft T cells and antigen-presenting cells (APC) [52-

55]. Donor-specific HLA antibodies may contribute to graft rejection not only by binding to graft cells and subsequent activation of complement, but also through binding of Fc receptors that may promote antigen uptake in APC and initiate antibody-dependent cellular cytotoxicity (ADCC) by NK cells [56].

IV. The HLA system

The major histocompatibility complex (MHC) on chromosome 6 in humans is a

complex 4 Mb genetic region including more than 200 genes and encoding the human

leukocyte antigens (HLA) [57]. HLA controls the activity of the immune system by

presenting self and non-self peptides to the immune cells of the host. HLA class I

antigens are found on all nucleated cells, while class II antigens are to be found

mainly on APC. The former presents peptides generated inside the cell following

digestion in the proteasome of for example viral antigens, while the latter presents

antigens taken up from outside the cell [58]. Peptide loaded class I antigens are

recognized mainly by the T cell receptor on CD8+ T cells and by the killer cell

immunoglobulin-like receptors (KIR) of NK cells, while peptide-bearing class II

antigens are recognized by the TCR mainly on CD4+ T cells [58]. The class III region

of the MHC complex encodes, among other proteins, cytokines (e.g. TNF-α) and

components of the complement system (C2, C4, factor B) [59]. The MHC class I region

carries three loci encoding the HLA-A, -B, and C antigens, which are all structurally

similar. HLA class I antigens are made up of a heavy alpha chain of 45kDa controlled

by a gene in the relevant MHC locus (Fig. 1). It is associated with a smaller chain of

12kDa called β2-microglobulin. In July of 2014 there were approximately 2,800, 3,500

and 2,300 distinct alleles of HLA-A, -B and -C respectively [60]. There are three

distinct HLA class II antigens, DR, DQ and DP, each composed of one α and one β

chain (Fig. 1). There are four different DRβ genes, DRβ1, DRβ3, DRβ4 and DRβ5 [61].

55]. Donor-specific HLA antibodies may contribute to graft rejection not only by binding to graft cells and subsequent activation of complement, but also through binding of Fc receptors that may promote antigen uptake in APC and initiate antibody-dependent cellular cytotoxicity (ADCC) by NK cells [56].

IV. The HLA system

The major histocompatibility complex (MHC) on chromosome 6 in humans is a complex 4 Mb genetic region including more than 200 genes and encoding the human leukocyte antigens (HLA) [57]. HLA controls the activity of the immune system by presenting self and non-self peptides to the immune cells of the host. HLA class I antigens are found on all nucleated cells, while class II antigens are to be found mainly on APC. The former presents peptides generated inside the cell following digestion in the proteasome of for example viral antigens, while the latter presents antigens taken up from outside the cell [58]. Peptide loaded class I antigens are recognized mainly by the T cell receptor on CD8+ T cells and by the killer cell immunoglobulin-like receptors (KIR) of NK cells, while peptide-bearing class II antigens are recognized by the TCR mainly on CD4+ T cells [58]. The class III region of the MHC complex encodes, among other proteins, cytokines (e.g. TNF-α) and components of the complement system (C2, C4, factor B) [59]. The MHC class I region carries three loci encoding the HLA-A, -B, and C antigens, which are all structurally similar. HLA class I antigens are made up of a heavy alpha chain of 45kDa controlled by a gene in the relevant MHC locus (Fig. 1). It is associated with a smaller chain of 12kDa called β2-microglobulin. In July of 2014 there were approximately 2,800, 3,500 and 2,300 distinct alleles of HLA-A, -B and -C respectively [60]. There are three distinct HLA class II antigens, DR, DQ and DP, each composed of one α and one β chain (Fig. 1). There are four different DRβ genes, DRβ1, DRβ3, DRβ4 and DRβ5 [61].

Fig. 1 The HLA class I and HLA Class II molecules

The strongest evidence that the HLA system is indeed the major histocompatibility system relevant for matching in transplantation comes from the fact that kidney or bone marrow grafts exchanged between HLA identical siblings survive almost as long as grafts between identical twins and far better than grafts exchanged between mismatched siblings or other relatives [62]. Advances in immunogenetics and histocompatibility testing have facilitated the clinical transplantation of solid organs and tissues. Improved definition of HLA antigens, alleles, and haplotypes has clarified the diversity of the HLA system among different racial/ethnic populations [63].

V. HLA antibodies

Antibodies, also called immunoglobulins, are large Y-shaped proteins, which function to identify and help remove foreign antigens or microbes such as viruses and bacteria (Fig. 2). Every different antibody recognizes a specific foreign antigen. This is because the two tips of its “Y” are specific to each antigen, allowing different antibodies to bind to different foreign antigens [64].

α2

2m α1

α3

1 α1

2 α2

HLA class I HLA class II

peptid

β

β

β

Fig. 2 Antibody structure

Antibodies are produced by the immune system in response to the presence of an antigen. Antigens can be carbohydrates like the blood group ABH antigens, lipids, e.g. phosphocholine or proteins. Antibodies are found circulating in blood, but are also present in the tissue interstitium and the various mucosae of the body [65]. There are five distinct classes of antibody namely IgG, IgA, IgM, IgD and IgE. They differ in size, charge, amino acid composition and carbohydrate content, but they share a similar basic structure [66]. The antibody molecule is bi-functional, the Fab component is used to bind antigen while the Fc region mediates the biological effect and is designated the effector region [67]. There are four IgG subclasses (IgG1, 2, 3, 4).

IgG1 and G3 are most efficient activators of complement. IgG and IgM have been shown to mediate graft rejection [68]. Interestingly, IgA HLA antibodies may have a protective effect [69, 70].

Antigen binding sites Variable region on heavy chain

Variable region on

light chain Fab

FC Disulfide bridges

Complement binding sites

Antigen binding by antibodies is their primary function and can result in protection of the host. Examples include neutralization of invading viruses or bacteria. Even though recent research has shown that binding of cell surface-expressed antigens, e.g.

HLA, can directly activate cells [71], the most significant biological effects mediated by antibodies are a consequence of their effector functions mediated by the Fc part [72]. Usually the ability to carry out a particular effector function requires that the antibody bind to its antigen. The effector functions include complement fixation and activation, and Fc receptor binding [72, 73].

The immune system responds to HLA antigens that are non-self. Healthy individuals may carry anti-HLA antibodies as a consequence of sensitization via pregnancies, blood transfusions or a previous transplant. It has been claimed that 15-25% of women develop HLA antibodies after their first pregnancy and 50-60% after their second pregnancy [74]. Likewise, the frequency of patients acquiring HLA antibodies following blood transfusions increase with the number of transfusions and may approach 70% in patients having had 20 transfusions and more [74]. With increasing number of mismatches between donor and patient in their first transplant, patients have higher PRA levels at relisting [75]. Thus, the benefits of better HLA matching at first transplant on lifetime with graft function are significant [75].

Patients with HLA antibodies and a high PRA value are less likely to receive a transplant because of a positive pre-transplantation crossmatch, and may also reject their grafts more readily even if the pre-transplantation crossmatch is negative [76].

As described above, donor-specific antibodies (DSAs) to HLA antigens can cause

acute AMR after kidney transplantation [75]. The clinical impact of HLA antibodies is

related to their antigen specificity, complement fixing ability, immunoglobulin class

and subclass, and titer [77]. Antibodies against HLA-A, -B, -Cw, -DRB1, -DRB3-5 and

Fig. 2 Antibody structure

Antibodies are produced by the immune system in response to the presence of an antigen. Antigens can be carbohydrates like the blood group ABH antigens, lipids, e.g. phosphocholine or proteins. Antibodies are found circulating in blood, but are also present in the tissue interstitium and the various mucosae of the body [65]. There are five distinct classes of antibody namely IgG, IgA, IgM, IgD and IgE. They differ in size, charge, amino acid composition and carbohydrate content, but they share a similar basic structure [66]. The antibody molecule is bi-functional, the Fab component is used to bind antigen while the Fc region mediates the biological effect and is designated the effector region [67]. There are four IgG subclasses (IgG1, 2, 3, 4).

IgG1 and G3 are most efficient activators of complement. IgG and IgM have been shown to mediate graft rejection [68]. Interestingly, IgA HLA antibodies may have a protective effect [69, 70].

Antigen binding sites Variable region on heavy chain

Variable region on

light chain Fab

FC Disulfide bridges

Complement binding sites

Antigen binding by antibodies is their primary function and can result in protection of the host. Examples include neutralization of invading viruses or bacteria. Even though recent research has shown that binding of cell surface-expressed antigens, e.g.

HLA, can directly activate cells [71], the most significant biological effects mediated by antibodies are a consequence of their effector functions mediated by the Fc part [72]. Usually the ability to carry out a particular effector function requires that the antibody bind to its antigen. The effector functions include complement fixation and activation, and Fc receptor binding [72, 73].

The immune system responds to HLA antigens that are non-self. Healthy individuals may carry anti-HLA antibodies as a consequence of sensitization via pregnancies, blood transfusions or a previous transplant. It has been claimed that 15-25% of women develop HLA antibodies after their first pregnancy and 50-60% after their second pregnancy [74]. Likewise, the frequency of patients acquiring HLA antibodies following blood transfusions increase with the number of transfusions and may approach 70% in patients having had 20 transfusions and more [74]. With increasing number of mismatches between donor and patient in their first transplant, patients have higher PRA levels at relisting [75]. Thus, the benefits of better HLA matching at first transplant on lifetime with graft function are significant [75].

Patients with HLA antibodies and a high PRA value are less likely to receive a transplant because of a positive pre-transplantation crossmatch, and may also reject their grafts more readily even if the pre-transplantation crossmatch is negative [76].

As described above, donor-specific antibodies (DSAs) to HLA antigens can cause

acute AMR after kidney transplantation [75]. The clinical impact of HLA antibodies is

related to their antigen specificity, complement fixing ability, immunoglobulin class

and subclass, and titer [77]. Antibodies against HLA-A, -B, -Cw, -DRB1, -DRB3-5 and

–DQB1 can all cause AMR, while more studies are needed in order to clearly link HLA-DPB and –DQA specific antibodies to AMR [77]. Interestingly, class II specific antibodies have been associated with the development of transplant glomerulopathy [78]. The ability of DSA to fix complement as inferred by C4d deposition in biopsies appears to be a poor prognostic marker for graft survival such that patients with DSA and C4d deposition in their cardiac grafts had worse graft survival than patients with DSA and no C4d deposition or patients without DSA but with C4d deposition [79].

The ability of preformed, low-level, DSA to trigger C4d fixation in vitro on single antigen beads in patients with negative conventional crossmatch tests is predictive for AMR. Assessment of C4d deposition on single antigen beads is potentially a powerful tool for risk stratification prior to transplantation and may allow identification of unacceptable donor antigens, or patients who may require enhanced immunosuppression [76]. Stastny et al have presented data suggesting that the donor- specific HLA antibodies of IgM type is predictive of transplant rejection in renal transplant recipients and susceptibility to coronary arteriopathy in heart grafts [80].

The most disputed topic in HLA antibody diagnostics relates to the mean fluorescence intensity (MFI) in the single antigen bead assay and what MFI is clinically significant. It has been suggested that the outcome of the single antigen bead assay should be interpreted in light of the crossmatch results, clinical outcome data and the clinical protocol of each center [77].

A number of programs and protocols have been implemented in order to make highly sensitized patients transplantable. They include the acceptable mismatch program of Eurotransplant, which is an algorithm that matches donor kidneys based on their HLA type and the HLA antibody repertoire of the patient [81]. In order to increase the availability of live donors to sensitized patients whose donor is

unacceptable because of DSA, paired kidney exchange programs have been established. Thus, two or more donor-recipient pairs can crosswise offer each other matched kidneys [82]. In addition, various desensitization protocols have been proposed that involves removal of DSA by plasmapheresis or immunoadsorption followed by prevention of the resynthesis of HLA antibodies by administration of intravenous immunoglobulins (IVIg) [83].

New treatment options such as eculizumab, an inhibitor of terminal complement activation, decreases the incidence of early AMR in sensitized renal transplant recipients (ClincalTrials.gov number NCT006707) [84].

VI. The complement system

Complement activation can occur in three ways by the classical pathway, the lectin

pathway, and by the alternative pathway. Both the classical and alternative pathways

converge on the activation of the C5 convertase, the activity of which results in the

production of C5b [85]. Antibody-mediated cytotoxicity is enhanced through

activation of complement via the classical complement pathway, which is initiated by

the binding of the C1q component to the Fc portion of IgM, IgG1 or IgG3. The final

product of complement activation, the membrane attack complex composed of C5b-

C9 subunits, creates a pore in the cell membrane resulting in cell lysis and death [86,

87]. Besides the presence in the circulation, complement factors such as C3 may be

produced locally in the graft, for example in tubular epithelial cells, and contribute to

both humoral and cellular rejection [88]. Complement activation is an important

contributor to AMR, and C4 deposition in the graft as revealed by histological

examination of a biopsy is an essential diagnostic criteria for AMR [89].